Expression of RAS and RAB interactor 1 (RIN1) in head and neck tumors at selected hospital in Ghana

Background Head and neck tumors (HNT) are tumors of the paranasal sinuses, the salivary glands and the upper aerodigestive tract. RIN1 is a Ras effector protein regulating epithelial cell properties and has been implicated in a number of cancers. Method The aim of this study was to investigate the expression of RIN1 in head and neck tumors. RIN1 expression was assessed using quantitative real-time PCR (qRT-PCR) and immunohistochemical staining on archival head and neck tissue samples between 2014 and 2020. Results RIN1 expression was low in tissue samples as compared with the normal head and neck tissues. High and low RIN1 levels were compared with ages ≤40, >40 in the head and neck tumors of p-value 0.02. There was a significant difference with p-values of 0.001 when poor and well-moderate malignant tumors were compared. Conclusion Our data suggests that RIN1may play an important role in head and neck tumor progression and that its expression may provide baseline data to facilitate identification of new molecular targeted therapeutics.

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ABSTRACT:
BACKGROUND: Head and neck tumor (HNT) are tumor of the paranasal sinuses, the salivary glands and the upper aerodigestive tract.RIN1 is a Ras effector protein regulating epithelial cell properties and has been implicated in a number of cancers.

METHOD:
The aim of this study was to investigate the expression of RIN1 in head and neck tumor.RIN1 expression was analyzed using quantitative real-time PCR (qRT-PCR) and immunohistochemical staining on tissue samples from a consecutive series of 150 head and neck tumor patients who underwent tumor resections between 2014 and 2020.

RESULTS:
The relationship between RIN1 expressions, clinicopathological factors, was investigated.qRT-PCR results showed that the RIN1 mRNA expression was low in tumor tissue samples than in t RIN1expression were low as compared with the normal head and neck tissues.High and low Rin 1 was compared with ages ≤40,>40 in the head and neck cancer of p-value 0.02.There was a significant difference between the histological differentiation of the malignant tumor with p values of 0.001, when poor and well moderate was compared.CONCLSION: Our data suggest that RIN1 plays an important role in head and neck tumor progression and that its expression will provide baseline data to facilitate identification of new molecular targeting therapeutics.

INTRODUCTION:
Head and neck cancers are cancers of the salivary glands, the paranasal sinuses (spaces around the nose lined with cells that make mucus), the nasal cavity, and the upper aerodigestive tract which includes the oral cavity, pharynx (throat), and larynx (voice box) with about 90% being squamous cell carcinomas (Kumar, et al, 2010;Pfeiffer et al., 2011).
The anatomical sites affected result in the loss of important functions such as speech, taste, smell and swallowing.
World Health Organization (WHO) in 2002 estimated an average of 600 000 new cases of HNC and 350 000 deaths each year worldwide.Oral cancer was identified as the commonest HNC with about 389 000 new cases annually followed by laryngeal and pharyngeal cancers with 160 000 and 65 000 new cases respectively.According to the WHO summary report update on human papillomavirus and related cancers in Ghana (September 15 2010), about three hundred and thirty (330) new cases of oral cancer are reported annually with approximately one hundred and fifty (150) deaths occurring out of this worldwide incident.
The development of HNC is a multistep process requiring the accumulation of genetic The RIN1 gene is located on chromosome 11q13.2(Shuster et al., 2000) and consists of a coding region of 2352bp constituting 783 amino acids.Its expression in cells is restricted, being predominantly expressed in forebrain neurons most notably in the hippocampus, cortex, striatum and amygdala (Dhaka et al., 2003).Moderate levels are expressed in mouse testes (Dhaka et al., 2003), hematopoietic cells, mammary epithelial cells and perhaps other epithelial cells of other tissues (Hu et al., 2005, Milstein et al., 2007).The expression level is however undetected in mouse embryonic fibroblast (Dzudzor et al., 2010), the midbrain and forebrain (Dhaka et al., 2003).The restricted expression is regulated by cis-acting elements located at the 5'region of the gene (Dzudzor et al., 2010).
RIN1 functions through two downstream pathways involved in maintenance of epithelial properties namely the activation of Rab5 (Tall et al., 2001) and Abl family tyrosine kinases (Hu et al., 2005).By activating ABL tyrosine kinases, RIN1 blocks the cytoskeletal rearrangements associated with cell dissociation and migration.The Ras and Rab interactor 1 (RIN1) is a multidomain Rab5 guanine nucleotide exchange factor that plays an important role in Rasactivated endocytosis and growth factor receptor trafficking (Tall et al., 2001).RIN1 is thus a Ras effector protein with its Ras binding domain (RBD) localized in its carboxyl terminal region.This region also binds 14-3-3 proteins which act as a negative regulator of membrane localization of RIN1 proteins (Wang et al., 2002).RIN1 interacts with the effector domain of activated Ras through competitive inhibition of Raf which is the best characterized of the Ras effector proteins.Raf is a mitogen activated protein kinase kinase kinase (MAP KKK) and its interaction with activated Ras gets it activated to set of the MAP kinase cascade which transduces signals resulting in changes in protein activity and gene expression.and lysosomes, an important cellular process known as receptor down-regulation for signal attenuation.Alterations in receptor trafficking and signal attenuation have been associated with carcinogenesis (Katzmann, et al 2002, Dikic and Giordano, 2003, Husnjak, and Dikic, 2006).
Of the many cellular systems that are impacted by EGFR signaling, the activation of the Rasdependent extracellular-regulated kinase (ERK) cascade appears to have the most pronounced effect on the proliferative response to epidermal growth factors (EGF).
While some of the genomic alterations correlate with genes known to be important in head and neck squamous cell carcinomas such as p16, p53, and Cyclin D1, many of the specific genes such as RIN1 are still unknown.This presents the need for research into possible genes such as RIN1 which encodes a protein regulating epithelial cell properties, and whose expression is found altered in cancerous breast and colorectal epithelial cells (Milstein et al., 2007, Senda et al, 2006).The purpose of this study is thus to provide useful information about the possible role of RIN1 in head and neck tumors and which may provide baseline data to facilitate the identification of new molecular targeting therapeutics.

Study Design
The study protocol was a retrospective in design, which consisted of laboratory analysis of
The relative levels of gene expression were represented as ΔCt = Ct of RIN1 -Ct of GAPDH, and the fold change of gene expression was computed using the 2-ΔΔCt method.

STATISTICAL ANALYSIS
All statistical analyses were performed using SPSS 17.0 for Windows (SPSS, Chicago, IL).
Significant level was set at 0.05.For qualitative variables, proportions and percentages was use to summarize the data.The association between RIN1 expression and HNT patient clinicopathologic features was evaluated using the χ2 test.107

RESULT THE LEVEL OF EXPRESSION OF RAS AND RAB INTERACTOR 1 (RIN1) IN HEAD AND NECK TUMOURS
Using logistic regression analysis, we determined the clinico-pathologic characteristics associated with high RIN1 expression in Head and Neck Malignant Tumor.Of the factors include in the analysis, only age was significantly associated with RIN1 expression.Patients with advanced age (>40 years old) presented with significantly lower odds ratio for expressing high RIN1 levels [OR = 0.34, 95% CI (0.13-0.85), p-value = 0.021] (Table 1.1).A similar risk stratification was performed for patients with benign Head and Neck Tumor.However, no statistically significant association between clinico-pathologic characteristics and relative expression of RIN1 was found (Table 1.2).

DISCUSSION
The Site of RIN1 in the Head and Neck Tumor Understanding the expression and the localization of RIN1 in head and neck tumor is of great value in developing a novel therapeutic strategy.RIN1 initiates Rab5 GTPases, which control endocytosis of cell-surface receptors, and also it initiates ABL non-receptor tyrosine kinases activities to participates in actin cytoskeleton remodeling.
RIN1 is involve in tumor metastasis and development but to our knowledge, there is no study on the level of RIN1 in head and neck tumors especially in Ghana.In this research, we investigated the level of expression of RIN1 in head and neck tumors and the normal tissues.
We also explored the correlation between the levels of RIN1 expression and clinical features such as gender, age and the anatomical site in head and neck tumor.
The RIN1 gene exhibits variable localization in different tumors.From this present study, RIN1 was localized at the cytoplasm in the head and neck tumor tissues (Micrograph 1.1 and 1.2), this is in accordance to a study by Senda et al (2007).From their study, RIN1 was found in the cytoplasm of colorectal cancer.The presence of RIN1 in the cytoplasm indicates that it is involved in the cell membrane signaling pathway to maintain the epithelial integrity (Milstein et al., 2007).RIN1 has the ability to inactivate uncontrolled cell growth which is initiated by Rab5 but this mechanism is inhibited when RIN1 is present at the cytoplasm (Pendergast, 2002).

The Level of Expression of RIN1 in Head and Neck Tumors and Normal Tissues
The level of expression of RIN 1 varies in tumors.From this present study, the level of expression of RIN1 is reduced or silenced in head and neck tumor tissues but was highly expressed in the normal head and neck tissues (Micrograph 1.1 and 1.2 and fig 1.1).This is similar to a study by Milstein et al (2007), they observed that the level of RIN1 was silence in breast tumor tissues but the level of expression of RIN1 increased in tumors such as gastric adenocarcinoma (Yu et al., 2012), colorectal cancer (Senda et al., 2007), non-small cell lung cancer (Wang et al., 2012), and bladder urothelial carcinoma (Shan et al., 2012) RIN1 is very crucial in the proliferative response to epidermal growth factors (EGF) and therefore low levels of RIN1 affects cell proliferation which is associated with the response to epidermal growth factor.The activation of Rab5 by RIN1 is therefore important in the control of the signaling pathway of EGFR.Therefore, when RIN1 is silenced, EGFR signaling will not be controlled by the Rab5 and this will result in a high abnormal proliferation which will favour tumorgenesis.
Patients with advanced age (>40 years old) presented with significantly lower odds ratio for expressing high RIN1 levels [OR = 0.34, 95% CI (0.13-0.85), p-value = 0.021] (Table 1.1).15 of the patients had poor differentiated tumor with low expression of rin1 out of 16 poor differentiated tumors (Table 1.1).There are two potential mechanisms that cause reduction in RIN1 expression.These mechanisms are DNA methylation of the RIN1 promoter and also transcriptional repression which result in high levels of SNAI1 (Barrallo et al., 2005).

The Effect of SNAI1 on RIN1 Expression
SNAI1 over expression contribute to RIN1 silencing in tumors.SNAI1 is a transcriptional repressor of genes that ensures the integrity of epithelial cell properties such as differentiation, cell division and cell to cell adhesion (Cano et al., 2000).Tumors with low RIN1 levels has a higher level of SNAI1 than their normal tissues and therefore elevated levels of SNAI1 contribute to the silencing of RIN1 in tumors (Marc et al., 2007).

The Effect of DNA Methylation on RIN1 Expression
The level of RIN1 expression is reduced as a result of DNA methylation.DNA methylation takes place at the CpG dinucleotides in the promoter region of RIN1 (Baylin et al., 2006).It therefore results in the mobilization of chromatin remodeling complexes in cells which causes a reduction in RIN1 expression (Baylin et al., 2006).

CONCLUSION
In summary, our results indicate that RIN1 is a tumor suppressor gene in HNT, making RIN1 an attractive target for future cancer therapeutics.
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by factors such as tobacco and alcohol intake, viral infections, poor nutrition and others against a background of heritable susceptibility to mutagens.
Rab5 proteins promotes endocytosis of EGFR and several other growth factor receptors.After activation, the receptor is degraded within multivesicular bodies (MVB) archived head and neck tumor tissue samples from 2014 to 2020.The data were accessed for the research purposes on 20 th March, 2021 from Cape Coast Teaching Hospital and Komfo Anokye Teaching Hospital.blocks of samples (archival tissue specimens) from patients with histologically HNT specimens were collected from the pathology laboratory at Cape Coast Teaching Hospital and komfo Anokye teaching Hospital in Ghana.Histologic diagnosis and differentiation were evaluated by a pathologist according to the World Health Organization classification system.The clinicopathologic features are shown in Table 1and 2. Prior to the initiation of this study, the research was approved by the Institutional Ethics Committee of the Cape Coast Teaching Hospital and the Committee of Human Research, Publications and Ethics at kwame Nkrumah University of Science and Technology, School of Medical Sciences & Komfo Anokye Teaching Hospital, the patients informed consent was waived by the ethics committees.The research protocol was approved by the Ethical Review Committee of the Cape Coast Teaching Hospital (REF: CCTHERC/RS/EC/2017/52) and the Committee on Human research, publications and Ethics of Kwame Nkrumah University of Science and Technology, School of Medical Sciences and Komfo Anokye Teaching Hospital (CHRPE/AP/609/18).IMMUNOHISTOCHEMISTRYThe expression of RIN1 was analyzed by immunohistochemistry (IHC).The goat anti-RIN1 (Biosynthesis Biotechnology Co LTD, Beijing China) antibody was used.4um formalin fixed paraffin embedded (FFPE) mounted sections were deparaffinized, rehydrated and rinsed three times in distilled water.After heat antigen retrieval and cooling in citrate buffer solution, they were incubated with 5% bovine serum albumin (BSA) at 37 o C for 30 minutes following three times phosphate-buffered saline (PBS) rinses.The slides were incubated at 4 o C with primary antibody RIN 1(1:500 dilution) overnight followed by incubation with Biotinylated anti-rabbit IgG) and SABC at 37 o C for 30 minutes each.The slides were thoroughly rinsed in between incubations with PBS three times.Next, they were developed for 5 minutes in 3,3'-diaminobenzene (DAB) and counter-stained with Mayer's hematoxylin at room temperature.Subsequently, they were washed with distilled water, HCL in ethanol and PBS.They were then incubated in PBS at 37 o C for 40 minutes to 1 hour.Finally, the slides were dehydrated in graded series of ethanol and xylene, cover slipped and mounted under the microscope.The brown and blue stained cells viewed under the microscope represent positive and negative cells respectively.The immunoreaction was subjectively assessed by experienced pathologists.RIN1-expressing cells were scored semiquantitatively according to the number of positive-staining cells and the staining intensity.cytoplasmic immunostaining in the tumor cells was considered positive staining.The immunohistochemical results of RIN1 were grouped into 2 categories: low expression (0 to 1+) and high expression (2+ to 3+).

Figure 1 .
Figure 1.1 shows the RIN1 expression levels in the normal head and neck tissues compared

Micrograph 1. 2
Immunohistochemistry expressions of RIN1 in tumour tissue.A is at tumour tissue with negative RIN1 expression (0); B is a tumour tissue with a weak expression (1+); C is a tumour tissue with moderate expression of RIN1 (2+); D is a tumour tissue with highly expression of RIN1 (3+).The data was produced from 3-5 fields per slide.Magnification ×400.Used arrows to point out the histological differences.

Figure 1 .
Figure 1.1 A graph showing the level of expression of RIN1 in the control and the tumour tissues by real-time polymerase chain reaction.